Aniridia due to a novel microdeletion affecting PAX6 regulatory enhancers: case report and review of the literature

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1 Journal of Genetics, Vol. 97, No. 2, June 2018, pp Indian Academy of Sciences RESEARCH NOTE Aniridia due to a novel microdeletion affecting PAX6 regulatory enhancers: case report and review of the literature ANDREAS SYRIMIS 1,NAYIANICOLAOU 1, ANGELOS ALEXANDROU 2, IOANNIS PAPAEVRIPIDOU 2, MICHAEL NICOLAOU 1, ELENI LOUKIANOU 3, VIOLETTA CHRISTOPHIDOU-ANASTASIADOU 1,4, STAVROS MALAS 5, CAROLINA SISMANI 2 and GEORGE A. TANTELES 1 1 Department of Clinical Genetics, The Cyprus Institute of Neurology and Genetics, 2370 Nicosia, Cyprus 2 Department of Cytogenetics and Genomics, The Cyprus Institute of Neurology and Genetics, 2370 Nicosia, Cyprus 3 Department of Ophthalmology, Nicosia General Hospital, 2370 Nicosia, Cyprus 4 Department of Clinical Genetics, Archbishop Makarios III Medical Centre, 2370 Nicosia, Cyprus 5 Department of Developmental and Functional Genetics, The Cyprus Institute of Neurology and Genetics, 2370 Nicosia, Cyprus *For correspondence. gtanteles@cing.ac.cy. Received 23 May 2017; revised 20 July 2017; accepted 28 August 2017; published online 25 May 2018 Abstract. Aniridia is a rare congenital ocular malformation that follows an autosomal dominant mode of inheritance. Most patients carry pathogenic point mutations in the paired box 6 gene (PAX6), but some carry deletions involving the 11p13 region, encompassing partly or completely PAX6 or the region downstream. We identified a novel deletion, 564 kb in size located about 46.5 kb downstream of PAX6 in a family with bilateral aniridia and foveal hypoplasia using array-cgh and multiplex ligation-dependent probe amplification. We also review all of the reported deletions downstream of PAX6 in patients with aniridia and/or other congenital malformations and define the overlapping region that leads to aniridia when deleted. Keywords. aniridia; array-cgh; multiplex ligation-dependent probe amplification; PAX6 deletion. Introduction Classic aniridia (MIM: ) is a rare congenital panocular malformation characterized by complete or partial iris hypoplasia due to heterozygous PAX6 mutations (Nelson et al. 1984; Samant et al. 2016). It can be accompanied by foveal hypoplasia, strabismus and optic nerve hypoplasia, generally leading to impaired visual acuity, while late-onset manifestations may include nystagmus, glaucoma, cataract and corneal abnormalities (Valenzuela and Cline 2004). It can occur either as an isolated malformation or as part of a syndrome, such as Wilms tumour, aniridia, genital anomalies, and mental retardation (WAGR, MIM: ), which is caused by 11p13 deletions involving PAX6 and the adjacent WT1 locus (Miller et al. 1964). Moreover, patients with aniridia may have nonocular sensory and neurological abnormalities, such as reduced olfaction and hearing difficulties (Hingorani et al. 2012). The prevalence of aniridia ranges from 1 in 50,000 to 1 in 100,000 live births (Gronskov et al. 2001). Approximately two thirds of all cases are familial following an autosomal dominant mode of inheritance with complete penetrance and variable expressivity, while the remaining cases are sporadic (Gronskov et al. 2001). Heterozygous loss-of-function mutations in PAX6 are identified in about 94% of patients with aniridia (Robinson et al. 2008). PAX6 encodes a transcription factor that plays a crucial role in early ocular morphogenesis and development of the central nervous system. It is expressed in nasal structures, gut, pancreas, pituitary, brain and spinal cord during embryonic development. It regulates the expression of other developmental regulatory genes, cell adhesion molecules, structural proteins and also of PAX6 itself (Simpson and Price 2002). The majority of PAX6 pathogenic mutations, including nonsense, splice-site and frame-shift mutations, cause the production of truncated transcripts resulting in haploinsufficiency. Some patients though carry deletions 555

2 556 Andreas Syrimis et al. Figure 1. Ophthalmological findings in the index patient and her son. (a) Slit-lamp photographs of the index patient (top) and her son (bottom) showing complete hypoplasia of the iris in both patients. Also lens dislocation in the left eye of the index patient is shown as well as opacities in both of her lenses. (b) The fundus photographs of the index patient (top) and her son (bottom) showing foveal hypoplasia. (c) The optical coherence tomography (OCT) of the macula confirming foveal hypoplasia in the index patient (left) and her son (right). L, left eye; R, right eye. at 11p13, which are thought to be rare causes of aniridia, leading to partial PAX6 deletion, completepax6 deletion, or deletion of the 3 PAX6 regulatory region leaving its entire coding region completely intact (Crolla and Heyningen 2002). In this study, we report the identification of a novel microdeletion affecting the 3 PAX6 regulatory region in a patient with familial aniridia and review the previously described phenotypes associated with similar microdeletions. Materials and methods Informed consent was obtained from all family members. Bidirectional Sanger sequencing of PAX6 was performed on an ABI 3130XL genetic analyzer (Applied Biosystems, Foster City, USA). Array-CGH analysis was performed using the Cytochip ISCA array (ver. 1.0, BlueGnome, Cambridge, UK) with 180,000 oligos in a 4x180k format. Fluorescent ratios were calculated using the Bluefuse Multi software (ver. 4.2, BlueGnome). Figure 2. (a) The array-cgh results showing a heterozygous deletion on chromosome 11p13 at base positions 31,195,401 and 31,722,881. This region encompasses the genes DCDC1, DNAJC24, IMMP1L and partially ELP4. The screenshot from the UCSC genome browser shows that the deletion spans three potentially active enhancers, as indicated by the high level of enrichment of the H3K27ac histone mark (circled). These also contain DNaseI hypersensitive sites, which also indicate the presence of regulatory regions as they tend to be DNase-sensitive, and transcription factor binding sites indicated by the black boxes. One of these regions also seems to be highly conserved as indicated by a very high level of evolutionary conservation across 100 vertebrate species. (b) Schematic presentation of the previously reported deletions at 11p13 including the one identified in our patient. Regulatory elements SIMO, HS2-HS3, HS5 and HS6 are indicated with blue vertical boxes. Horizontal bars represent deletions that have been identified in patients with aniridia and/or other ocular malformations (red), neurodevelopmental disorders in addition to aniridia and/or other ocular malformations (blue) and neurodevelopmental disorders without ocular malformations (yellow). The vertical dashed lines indicate the overlapping region that is deleted in all patients with complete aniridia, referred to as the critical region (chr11: 31,422,424 31,666,340). 1*, ocular coloboma phenotype; 2*, partial aniridia phenotype.

3 Novel 11p13 microdeletion 557.

4 558 Andreas Syrimis et al. Multiplex ligation-dependent probe amplification (MLPA) was conducted using the SALSA probemix P219-B3 (MRC-Holland, Amsterdam, The Netherlands). Fragment separation by capillary electrophoresis was performed on an ABI 3130xl genetic analyzer. MLPA analysis was performed using the Coffalyser.Net software (ver. 1.4, MRC-Holland). Quantitative real time PCR was carried out on the CFX96 Real-Time C1000 Thermal Cycler (Biorad, Hercules, USA) using the SsoFast Evagreen Supermix (Biorad). Several primers were initially designed to span the region adjacent to the deletion breakpoints at intervals of 5000 bp and then new primers were designed at 700 bp intervals. Data analysis was performed using the CFX Manager Software (Biorad). Results A 44-year-old female was referred to the Clinical Genetics clinic with the diagnosis of aniridia. Her 13-year-old son was also affected. Review of her family history revealed that she was one of the four siblings born to nonconsanguineous parents. One of her maternal uncles was diagnosed with renal cancer at the age of 50 years. Family history was otherwise noncontributory for aniridia. The proband was an IUGR baby born close to term with a birth weight of 1.9 kg. Bilateral aniridia was identified in early infancy. Her development was unremarkable. At the age of 35, she was incidentally diagnosed with type 2 diabetes mellitus. At the age of 38, she underwent surgery for a multinodular goiter. Histology revealed an 8-mm papillary thyroid carcinoma of the isthmus (T1N0M0). She developed postural tremor at the age of 42. She also had a history of hyperlipidaemia and hypovitaminosis D. On examination, her occipitofrontal circumference (ofc) was 55 cm (50th 75th centile), her weight was 79 kg (91st 98th centile) and her height was 154 cm (2nd 9th centile). She was obese with a BMI 33.3 kg/m 2. She was facially nondysmorphic. She had a short neck. She had upper chest and shoulder freckling. She had a 2/6 soft systolic heart murmur. Neurology examination was nonfocal. On ophthalmology evaluation, she had horizontal nystagmus and uncorrected visual acuity of 0.2 in each eye, which increased to 0.4 when corrected with myopic lenses. The intraocular pressure was 20 mmhg in the right eye and 21 mmhg in the left eye. Slit lamp examination showed near total aniridia in both eyes (figure 1a). The intraocular lens in the left eye was superiorly subluxated with stretched zonules and with opacities in both eyes (cataract). Gonioscopy showed rudimentary iris tissue. Fundoscopy revealed grade 3 foveal hypoplasia, which was confirmed by optical coherence tomography (OCT) of the macula (figure 1, b c). Examination of her son at the age of 13 years and 10 months revealed an ofc of 55.5 cm (25th 50th centile), a weight of 76 kg (98th 99.6th centile) and height of 166 cm (50th 75th centile). He had bilateral aniridia. He had a single left-sided irregular hyperpigmented macule over the upper abdomen. Cardiovascular examination revealed a 2 3/6 systolic heart murmur. He was overweight with a BMI of 27.6 kg/m 2. Neurology examination was nonfocal. Abdominal ultrasound scan was unremarkable. He had horizontal nystagmus and uncorrected visual acuity of 0.5 in each eye, which increased to 0.9 with hypermetropic lenses. The intraocular pressure was 16 mmhg in the right eye and 17 mmhg in the left eye. Slit lamp examination showed near total aniridia in both eyes (figure 1a). The intraocular lens was clear without any opacities and without any lens dislocation in either eye. Fundoscopy revealed grade 1 foveal hypoplasia without any other retinal abnormality, which was confirmed with OCT of the macula (figure 1, b c). Abdominal ultrasound scan was unremarkable. Bidirectional Sanger sequencing of the coding and flanking regions of PAX6 revealed no mutations in the proband and her son. Array-CGH detected a novel heterozygous 3 deletion of 577 kb in size located 37-kb downstream of PAX6 in the 11p13 region (chr11: 31,195,401 31,772,881; figure 2a). This finding was confirmed by MLPA in both proband and her son. Subsequently, qrt-pcr refined the deletion size to about 564 kb located 46.5-kb downstream of PAX6 (chr11: 31,200,388 31,764,028), encompassing the genes DCDC1, DNAJC24, IMMP1L and partially ELP4. This region also encompasses three sites that show enrichment for the H3K27ac histone mark, which indicates the presence of potentially active enhancers. In addition, one of these sites that show enrichment for the H3K27ac mark also show a high level of evolutionary conservation across 100 vertebrate species, further supporting the presence of regulatory elements (figure 2b). Discussion and review of the literature In this study, we identified a novel 564 kb deletion downstream of PAX6 in a family with bilateral aniridia and foveal hypoplasia. Deletions of the 3 regulatory regions downstream of PAX6 abrogate its expression leading to aniridia due to PAX6 haploinsufficiency, a phenomenon known as position effect (Fantes et al. 1995). Twenty patients with a similar deletion downstream of PAX6 were reported in the literature to date. The phenotypes of these patients, the breakpoints as well as the genes within the deleted region are summarized in table 1. All these patients had aniridia, except a patient with a deletion 114-kb downstream of PAX6, who had ocular coloboma (Guo et al. 2013). A patient with a larger deletion (1.3 Mb) encompassing MPPED2,, DCD1, IMMP1L, DNAJC24 and ELP4 was also reported to have autism spectrum disorder and intellectual disability in addition to aniridia (Davis et al. 2008).

5 Novel 11p13 microdeletion 559 Table 1. Deletions identified downstream of PAX6previously reported in literature. Distance from the 3 of PAX6 (kb) Location (hg19) Deleted genes (whole or partially) Size (kb) Phenotype Aniridia and/or other ocular malformations Lauderdale et al. (2000) 22.1 Unknown ELP4, IMMP1L, DNAJC24, DCDC1, 11.6 Unknown ELP4, IMMP1L, DNAJC24, DCDC1, 975 Aniridia 1105 Aniridia Bayrakli et al. (2009) 140 chr11:31,260,340 31,666,340 ELP4, IMMP1L, DNAJC24, DCDC1 406 Aniridia Zhang et al. (2011) 1 chr11:31, 280,628 31,805,329 ELP4, IMMP1L, DNAJC24, DCDC1 525 Aniridia Cheng et al. (2011) 123 chr11:31,117,827 31,683,687 ELP4, IMMP1L, DNAJC24, DCDC1 566 Aniridia Wawrocka et al. (2012) 85 chr11:31,122,161 31,721,030 ELP4, IMMP1L, DNAJC24, DCDC Partial aniridia Guo et al. (2013) 114 chr11:31,010,914 31,692,238 ELP4, IMMP1L, DNAJC24, DCDC1, 681 Ocular coloboma Addis et al. (2015) 96 chr11:31,118,027 31,710,576 ELP4, IMMP1L, DNAJC24, DCDC1 593 Rieger anomaly, aniridia 31 chr11:31,172,410 31,775,457 ELP4, IMMP1L, DNAJC24, DCDC1 603 Aniridia 23 chr:31,605,859 31,783,590 ELP4 178 Partial aniridia Ansari et al. (2016) 108 chr11:30,918,066 31,698,257 ELP4, IMMP1L, DNAJC24, DCDC1, Blanco-Kelly et al. (2017) 59 chr11:31,010,424 31,747,424 ELP4, IMMP1L, DNAJC24, DCDC1, 780 Aniridia 737 Aniridia 113 chr11:31,152,003 31,693,266 ELP4, IMMP1L, DNAJC24, DCDC1 541 Aniridia 54 chr11:31,422,424 31,751,424 ELP4, IMMP1L, DNAJC Aniridia 91 chr11:31,147,306 31,714,853 ELP4, IMMP1L, DNAJC24, DCDC1 567 Aniridia 108 chr11:31,186,493 31,698,208 ELP4, IMMP1L, DNAJC24, DCDC1 512 Aniridia 101 chr11:31,083,877 31,704,548 ELP4, IMMP1L, DNAJC24, DCDC1 620 Aniridia Aniridia and/or other ocular malformations and neurodevelopmental disorders Davis et al. (2008) 4 chr11:30,448,178 31,802,357 ELP4, IMMP1L, DNAJC24, DCDC1,, MPPED2 Addis et al. (2015) 242 chr11:30,991,456 31,564,708 ELP4, IMMP1L, DNAJC24, DCDC1, 1354 Aniridia, autism, moderate mental retardation 573 Focal epilepsy with cortical dysplasia, mild developmental delay, adhd, neurinomas, squint, ptosis, fine motor dyspaxia

6 560 Andreas Syrimis et al. Table 1 (contd) Distance from the 3 of PAX6 (kb) Location (hg19) Deleted genes (whole or partially) Size (kb) Phenotype Ansari et al. (2016) 11 chr11:31,277,819 31,795,239 ELP4, IMMP1L, DNAJC24, DCDC1 517 Partial aniridia, ataxia, developmental delay Neurodevelopmental disorders without aniridia Addis et al. (2015) 260 chr11:31,495,260 31,546,276 ELP4, IMMP1L 51 Cognitive delay, speech and language disorder, reading and spelling disorder, asd, epilepsy 181 chr11:31,561,220 31,625,448 ELP4 64 Speech and language delay 132 chr11:31,573,422 31,674,789 ELP4 101 Developmental delay, microcephaly 164 chr11:31,584,329 31,642,325 ELP4 58 Language disorder, behavior problems 174 chr11:31,601,768 31,632,347 ELP4 31 Developmental delay, hypotonia, ventriculomegaly 84 chr11:31,691,270 31,722,740 ELP4 31 Developmental delay, speech and language disorder, microcephaly, mild cognitive delay, motor skills development disorder 59 chr11:31,705,076-31,747,631 ELP4 43 Autism, learning difficulties 20 chr11:31,760,904 31,786,914 ELP4 26 Moderate developmental delay, autistic traits 4 chr11:31,597,322 31,802,120 ELP4 205 Severe intellectual disability, muscle hypotrophy, severe dysphagia, craniofacial abnormalities 31 chr11:31,605,859 31,775,457 ELP4 170 Developmental delay, behavioral disturbances, pervasive developmental disorder 31 chr11:31,625,389-31,775,457 ELP4 150 Behavioral and speech disorders, mild mental retardation 151 chr11:31,460,506 31,655,108 ELP4, IMMP1L 195 Autism 199 chr11:31,488,890 31,607,986 ELP4, IMMP1L 119 Autism 157 chr11:31,518,924 31,649,475 ELP4, IMMP1L 131 Autism, language delay 76 chr11:31,576,768 31,653,568 ELP4 77 Autism, coordination problems 112 chr11:31,652,219 31,764,393 ELP4 112 Autism, language delay, mild developmental delay, motor delay Balay et al. (2016) 191 chr11:31,452,082 31,615,319 ELP4, IMMP1L, DNAJC Autism, intellectual disability, speech abnormalities

7 Novel 11p13 microdeletion 561 To further assess the prevalence of our identified deletion, we searched the DECIPHER database for patients with similar deletions downstream of PAX6, yielding 12 additional aniridia patients (Firth et al. 2009). One of these patients in addition was reported to have congenital cataracts and nystagmus, while another one was also described to have global developmental delay. Another patient reported in DECIPHER had Rieger anomaly. Therefore, the prevalence of 11p13 microdeletion in affected individuals seems to be high, although clinical manifestations may vary depending on the size of the deletion, the location of the breakpoints and the genes involved. All deletions downstream of PAX6 previously reported to cause either aniridia or other ocular malformations, including the one identified in our study, encompass a common overlapping region 244 kb in size (chr11: 31,422,424 31,666,340; figure 2b). This was previously characterized by Ansari et al. (2016) as the critical region for aniridia. This region partially encompasses DNAJC24, IMMP1L and introns 1 7 of ELP4. A deletion including only part of the critical region was found in a patient with partial aniridia, suggesting that a partial deletion of this critical region possibly leads to a milder ocular phenotype (figure 2b) (Addis et al. 2015). Microdeletions including or disrupting ELP4 were also shown to be significantly enriched in patients with a range of neurodevelopmental disorders, including autism spectrum disorder, language impairment, epilepsy, developmental delay and intellectual disability (Addis et al. 2015). The phenotypes of these patients, the breakpoints as well as the genes within the deleted region are also summarized in table 1. These microdeletions do not seem to involve the entire critical region (figure 2b). Only three patients carrying a deletion downstream of PAX6 presented with aniridia (or other ocular malformations) in conjunction with a neurodevelopmental disorder (figure 2b) and two of these patients had a deletion involving the entire critical region. This indicates that deletions of the entire critical region (chr11: 31,422,424 31,666,340) cause aniridia or other ocular malformations with complete penetrance. Such deletions or smaller ones may also predispose patients to neurodevelopmental disorders with incomplete penetrance. There was no evidence of a neurodevelopmental disorder in our patients further supporting a complex genotype phenotype relationship. Genetic and functional studies support the presence of a PAX6 downstream regulatory region that is essential for PAX6 expression in the developing ocular tissues and causes aniridia when deleted. The downstream regulatory region contains six known regulatory elements: a lens-specific enhancer, an ultra-conserved cis-regulatory enhancer called SIMO, a retina-specific enhancer located in a fragment containing HS2 and HS3 and the recently identified HS5 and HS6 elements (Kleinjan et al. 2006; McBride et al. 2011). These elements are located in the introns 7 9 of ELP4, which are upstream of the critical region, suggesting that additional regulatory elements, essential for PAX6 expression likely exist within the critical region. Deletion of the entire downstream regulatory region in mice was previously shown to abrogate expression of Pax6 in the retina, iris and the ciliary body (Kleinjan et al. 2006). This indicates that the downstream regulatory region is essential for PAX6 expression providing additional evidence to support that the underlying genetic cause of aniridia in our patients is the identified deletion, which spans the downstream regulatory region and thereby abolishes PAX6 expression in the developing eye. In conclusion, we have identified a novel 564 kb microdeletion downstream of PAX6 in a family with bilateral aniridia and foveal hypoplasia using both array-cgh and MLPA. The phenotype of this patient shows a great overlap with that of previously reported patients who have a similar deletion. The use of array-cgh or MLPA of PAX6 and downstream regulatory regions is highly recommended for aniridia patients in conjunction with PAX6 mutation screening, to facilitate the ascertainment of risk for other conditions, since ELP4 and WT1 deletions have been associated with neurodevelopmental disorders and Wilms tumour, respectively. Acknolwedgement We thank the family members for participating in this study. References Addis L., Ahn J. W., Dobson R., Dixit A., Ogilvie C. M., Pinto D. et al Microdeletions of ELP4 are associated with language impairment, autism spectrum disorder, and mental retardation. Hum. Mutat. 36, Ansari M., Rainger J., Hanson I. M., Williamson K. A., Sharkey F., Harewood L. et al Genetic snalysis of PAX6-negative individuals with aniridia or Gillespie syndrome. PLoS One 11, e Balay L., Totten E., Okada L., Zell S., Ticho B., Israel J. et al A familial pericentric inversion of chromosome 11 associated with a microdeletion of 163 kb and microduplication of 288 kb at 11p13 and 11q22.3 without aniridia or eye anomalies. Am. J. Med. Genet. A 170A, Bayrakli F., Guney I., Bayri Y., Ercan-Sencicek A. G., Ceyhan D., Cankaya T. et al A novel heterozygous deletion within the 3 region of the PAX6 gene causing isolated aniridia in a large family group. J. Clin. Neurosci. 16, Blanco-Kelly F., Palomares M., Vallespin E., Villaverde C., Martin-Arenas R., Velez-Monsalve C. et al Improving molecular diagnosis of aniridia and WAGR syndrome using customized targeted array-based CGH. PLoS One 12, e Cheng F., Song W., Kang Y., Yu S. and Yuan H A 556 kb deletion in the downstream region of the PAX6 gene causes familial aniridia and other eye anomalies in a Chinese family. Mol. Vis. 17,

8 562 Andreas Syrimis et al. Crolla J. A. and van Heyningen V Frequent chromosome aberrations revealed by molecular cytogenetic studies in patients with aniridia. Am. J. Hum. Genet. 71, Davis L. K., Meyer K. J., Rudd D. S., Librant A. L., Epping E. A.,SheffieldV.C.et al Pax6 3 deletion results in aniridia, autism and mental retardation. Hum. Genet. 123, Fantes J., Redeker B., Breen M., Boyle S., Brown J., Fletcher J. et al Aniridia-associated cytogenetic rearrangements suggest that a position effect may cause the mutant phenotype. Hum. Mol. Genet. 4, Firth H. V., Richards S. M., Bevan A. P., Clayton S., Corpas M., Rajan D. et al DECIPHER: database of chromosomal imbalance and phenotype in humans dsing ensembl resources. Am. J. Hum. Genet. 84, Gronskov K., Olsen J. H., Sand A., Pedersen W., Carlsen N., Bak Jylling A. M. et al Population-based risk estimates of Wilms tumor in sporadic aniridia. A comprehensive mutation screening procedure of PAX6 identifies 80% of mutations in aniridia. Hum. Genet. 109, Guo H., Dai L., Huang Y., Liao Q. and Bai Y A large novel deletion downstream of PAX6 gene in a Chinese family with ocular coloboma. PLoS One 8, e Hingorani M., Hanson I. and van Heyningen V Aniridia. Eur. J. Hum. Genet. 20, Kleinjan D. A., Seawright A., Mella S., Carr C. B., Tyas D. A., Simpson T. I. et al Long-range downstream enhancers are essential for Pax6 expression. Dev. Biol. 299, Lauderdale J. D., Wilensky J. S., Oliver E. R., Walton D. S. and Glaser T deletions cause aniridia by preventing PAX6 gene expression. Proc. Natl. Acad. Sci. USA 97, McBride D. J., Buckle A., van Heyningen V. and Kleinjan D. A DNaseI hypersensitivity and ultraconservation reveal novel, interdependent long-range enhancers at the complex Pax6 cis-regulatory region. PLoS One 6, e Miller R. W., Fraumeni J. F., Jr. and Manning M. D Association of Wilms s tumor with aniridia, hemihypertrophy and other congenital malformations. N. Engl. J. Med. 270, Nelson L. B., Spaeth G. L., Nowinski T. S., Margo C. E. and Jackson L Aniridia. A review. Surv. Ophthalmol. 28, Robinson D. O., Howarth R. J., Williamson K. A., van Heyningen V., Beal S. J. and Crolla J. A Genetic analysis of chromosome 11p13 and the PAX6 gene in a series of 125 cases referred with aniridia. Am. J. Med. Genet. A 146A, Samant M., Chauhan B. K., Lathrop K. L. and Nischal K. K Congenital aniridia: etiology, manifestations and management. Expert Rev. Ophthalmol. 11, Simpson T. I. and Price D. J Pax6; a pleiotropic player in development. BioEssays 24, Valenzuela A. and Cline R. A Ocular and nonocular findings in patients with aniridia. Can. J. Ophthalmol. 39, Wawrocka A., Budny B., Debicki S., Jamsheer A., Sowinska A. and Krawczynski M. R PAX6 3 deletion in a family with aniridia. Ophthalmic Genet. 33, Zhang X., Zhang Q., Tong Y., Dai H., Zhao X., Bai F. et al Large novel deletions detected in Chinese families with aniridia: correlation between genotype and phenotype. Mol. Vis. 17, Corresponding editor: Rajiva Raman